Application
Western blot
Sample
Mouse Tissue lysate - whole (brain sample)
Gel Running Conditions
Reduced Denaturing (gel 10%)
Loading amount
10 µg
Specification
brain sample
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Other product details
Incubation time
12 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: saturation buffer + tween 20 (0.1%)
Dilution
1/1000
Secondary antibody
Dilution
1/5000
Name
Non-Abcam antibody was used: Amersham Biosciences, ECL Anti-Rabbit IgG
Host species: Donkey
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Host species: Donkey
Clonality: Polyclonal
Conjugation: Horse Radish Peroxidase
Detection
Detection method
ECL+
Exposure
2 minute(s) and 0 second(s)
Positive control
Positive controls were made after stripping.
First positive control was made with an Ab directed against the loading protein beta-tubulin.
A second control was made with the anti-beta2 anibody from Genetex (GTX113654) which showed bands at the match weigh, but non specific however (immunoreactivity in knock-out sample).
Additional data
Additional Notes
Sample were prepared from fresh brain samples.
Lysis was made with 10 mM Tris-HCl, 50 mM NaCl, 1% Triton X-100, 30 mM sodium pyrophosphate, 50 mM NaF, 5 µM ZnCl2, 100 µM Na3PO4, 1 mM DTT, 5 nM okadaic acid, 2.5 µg aprotinin, 2.5 µg pepstatine, 0.5 µg PMSF, 0.5 mM benzamidine, 2.5 µg leupeptin.
Samples were denaturated at 95°C for 5 min.
PVDF membrane were used.
On the scan, you could see 3blots.
First is the blot with Ab41174 (1/500e)
2nd is with beta-tubulin antibody (1/1000e)
3rd is with the GeneTex anti-beta2 Ab (1/500e).
Conditions are the same for the 3blots, with the first lane as the weigh marker, 2 and 3 with sample from 4month-old mice, 4 and 5 with sample from 18month-old mice, 6 and 7 with sample from knock-out beta2 mice.
We tried several conditions (blocking with BSA, primary and secondary antibody dilution, exposure time, etc.), and nonetheless we obtained a whole fluorescent membrane without any bands.
Abcam response
Thank you for your honest feedback.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Mr. M Vandesquille
Verified customer
提交于 Dec 02 2010